The Principal Investigator's long term research objectives are to elucidate the role of sterol carrier protein-2 (SCP2) in hepatocellular trafficking and maintenance of cellular unesterified cholesterol levels. Ultimately, this information will be applicable to the study of a variety of disease states associated with derangements in these processes, particularly atherosclerosis. Current knowledge of cellular cholesterol metabolism suggests that SCP2, a well characterized in vitro cholesterol transfer protein, may be important in mediating the intermembrane and intramembrane transfer of cholesterol. This has important implications for the regulation of both cholesterol synthesis and metabolism. At present little is known about the in vivo function of this protein, however, the limited data available suggests a complex pattern of tissue specific expression and regulation which suggests that SCP2 may have different functions depending on the cell type examined. In the liver, SCP2 is a highly expressed peroxisomal protein which is regulated in association with growth specific cues. We postulate that SCP2 may function as a substrate dependant cofactor in cholesterol and bile acid synthesis and in new membrane formation during periods of cell growth. The specific aims of this study are: (1). To elucidate the mechanisms involved in the regulation of cellular levels of SCP2; (2). To inhibit SCP2 expression using antisense oligonucleotides and to correlate this with changes in cholesterol synthesis and plasma membrane cholesterol levels in growth stimulated cells; and (3). To overexpress SCP2 using an eukaryotic expression vector-SCP2 cDNA construct and measure the influence of this change on cholesterol metabolism. For these studies we will utilize Reuber H-35 rat hepatoma cells which are well differentiated and retain many of the synthetic functions of normal rat liver, prominent among which is the synthesis of bile acids. Modulation of SCP2 levels by growth factors and peroxisomal proliferators will be examined to determine the translational and post-translational processes involved in the regulation of SCP2 levels. The role of SCP2 in hepatocellular cholesterol and bile acid synthesis and in new membrane formation will be assessed by correlating changes in these events with alterations in the level of SCP2 expression. Successful completion of these studies will provide important insights into the role of this protein in cellular cholesterol metabolism.